Author ORCID Identifier

http://orcid.org/0000-0001-6844-3367

Date Available

12-23-2016

Year of Publication

2016

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Pharmacy

Department/School/Program

Pharmaceutical Sciences

First Advisor

Dr. Wooin Lee

Second Advisor

Dr. Kyung Bo Kim

Abstract

Proteasome inhibitors (PIs) are a class of FDA-approved anti-cancer agents which includes the first-generation PI bortezomib (BTZ) and second-generation carfilzomib (CFZ). Drug resistance is a major challenge in PI therapy with no solution currently available. While a few resistance mechanisms had been proposed for BTZ, little was known about CFZ resistance before the start of our studies. In this dissertation work, we investigated multiple mechanisms contributing to CFZ resistance—alterations in the drug transporter activity, metabolic stability, and proteasome activity profiles—and evaluated potential strategies to overcome CFZ resistance.

We observed marked upregulation of the drug efflux transporter P-glycoprotein (P-gp) in our H23 (lung cancer) and DLD-1 (colorectal cancer) cell line models of acquired resistance. P-gp inhibition by verapamil effectively restored CFZ sensitivity in resistant cells, indicating that P-gp contributes to CFZ resistance in our model. We designed a small library of CFZ analogs lacking the pharmacophore and screened them for their abilities to reverse CFZ resistance. Our results showed that dipeptide CFZ analogs were the most effective in restoring CFZ sensitivity. This study was among the first to demonstrate the involvement of P-gp upregulation in CFZ resistance and the feasibility of using CFZ peptide analogs to reverse P-gp-mediated CFZ resistance.

PI-resistant cancer cells often exhibit altered proteasome activity profiles compared to PI-sensitive cells. To further explore how these changes to the proteasome may influence cellular response to PIs, we developed a pancreatic cancer cell line model of acquired CFZ resistance. CFZ-resistant BxPC3 cells displayed a marked increase in the caspase-like (C-L) activity of the proteasome compared to parental controls. When challenged with CFZ, we also found that C-L activity was preserved in resistant cells whereas all activities were inhibited in parental cells. Using both chemical and genetic knockdown approaches, we found that co-inhibition of the C-L activity can sensitize resistant cells to CFZ. Similar effects were also observed in CFZ-resistant RPMI-8226 multiple myeloma cells. These findings suggest that enhanced C-L activity may contribute to CFZ resistance and that combined inhibition of the C-L activity may serve as a potential strategy to restore CFZ sensitivity.

Since CFZ contains a tetrapeptide backbone and a highly reactive epoxyketone pharmacophore, its rapid metabolic inactivation in vivo may be a potential explanation for its lack of anti-cancer activity in solid cancers. Thus, we hypothesized that improving the metabolic stability of CFZ and its access to cancer cells may enhance its anti-cancer efficacy. Using micelle particles composed of biodegradable block copolymers poly-(ethylene glycol) (PEG) and poly-(caprolactone) (PCL), we demonstrated as a proof-of-concept that extended-release nanoformulations improved the metabolic stability and cytotoxic activity of CFZ in solid cancer cell lines. These findings supported the potential utility of polymer micelle formulations in enhancing the delivery of CFZ and improving anti-cancer efficacy CFZ against solid cancers.

Findings from this dissertation work enhance our understanding of factors contributing to CFZ resistance in cancer cells. Such information may be useful for the development of next-generation proteasome inhibitors and new strategies to combat CFZ resistance in the clinic.

Digital Object Identifier (DOI)

https://doi.org/10.13023/ETD.2017.004

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